Many aspects of gene function are coordinated by changes in the epigenome, which include dynamic revisions of chromatin modifications, genome packaging, subnuclear localization, and chromosome conformation. All of these epigenetic mechanisms are used by developing lymphocytes to regulate the production of functional antigen receptor genes by V(D)J recombination. This process of genetic rearrangement fuses randomly selected combinations of immunoglobulin (Ig) or T cell receptor (Tcr) gene segments to form a signature variable region exon for each precursor B or T cell, respectively. Although V(D)J recombination is critical for the production of an adaptive immune system, genetic reorganization is risky. Aberrant targeting of V(D)J recombination can produce chromosomal translocations involving proto-oncogenes. These translocations are initiating lesions in the vast majority of leukemias and lymphomas. As such, stringent control mechanisms ensure the stage-, tissue-, and allele-specific targeting of the recombination apparatus (V(D)J recombinase) during lymphocyte development. On a more localized level, V(D)J recombination is regulated by a complex interplay between genetic control elements that modify the accessibility of chromatin at Ig and Tcr loci. At a broader level, V(D)J recombination is controlled by active contraction of Ig and Tcr loci to bring distant gene segments into spatial proximity. The applicant's laboratory has shown that transcriptional promoters and enhancers serve as accessibility control elements (ACEs) within Ig heavy chain (Igh) and Tcrb loci. At the Tcrb locus, a stable structure forms between distant promoter and enhancer elements to recruit proteins that covalently modify and remodel nucleosomes, thus generating recombinase accessible chromatin. In contrast to our detailed understanding of recombinase targeting at Tcrb, little is known about the genetic and epigenetic mechanisms controlling Igh gene assembly, especially those that facilitate long-range rearrangement between VH gene segments and rearranged DHJH elements, located up to 2.5 Mb away. The central hypothesis of the proposed research project is that a series of known and novel genetic control elements mediate the opening of VH chromatin and subsequent interactions between this large cluster of gene segments and the distant DHJH region. To test this hypothesis, the applicant proposes to (i) determine whether known regulatory elements in the DHJH region are essential for Igh locus contraction in primary B cell precursors and (ii) identify novel genetic control elements within the VH cluster that activate transcription of its composite gene segments and coordinate their long-range interactions with the DHJH region. Together, these studies will provide a new paradigm for the chromosomal dynamics that guide antigen receptor gene assembly and prevent aberrant interactions that may lead to oncogenic translocations with other chromosomes. PUBLIC HEALTH RELEVANCE: The packaging and spatial organization of DNA in the nucleus are key determinants of gene function;changes in these "epigenetic" programs may be at the heart of aging and disease susceptibilities. In addition, epigenetic control is essential for the proper generation of antigen receptor genes because defects in this assembly process cause chromosomal translocations that lead to lymphoid tumors. We propose studies to address how precursor B cells control long-range changes in the spatial organization of the immunoglobulin heavy chain locus to ensure its proper assembly and pursuant generation of an adaptive immune system.